Xenotropic murine leukemia virus-related virus (XMRV) is a recently discovered human gammaretrovirus that resembles a xenotropic MLV, but that is distinguishable from xenotropic MLV in the sequence in its envelope (Urisman et al., PLOS pathogens 2(3):e25, 2006; and Dong et al. PNAS 104:1655, 2007). All isolates so far examined are highly homologous to each other (>98% sequence identity) and allow the distinction from xenotropic MLV. The reason for this sequence conservation is not currently understood. The original infectious clone is called XMRV VP62 (GenBank accession no. EF185282).
XMRV was originally described in association with prostate cancer and further connections have been suggested (R. Schlaberg et al. PNAS 2009, doi_10.1073_pnas.0906922106), with 6-23% of prostate cancer patients testing positive. In addition, V. C. Lombardi et al. (Science 2009 doi10.1126/science. 1179052) showed a possible association with chronic fatigue syndrome, with 67% of patients testing positive, compared to 3.7% of normals. Overall estimates of the prevalence in the general population from investigators in the USA range from 2-4%. However several recent studies in Europe have failed to detect XMRV in similar frequencies or similar associations (Fischer et al. Journal of Clinical Virology 43: 277-283 2008; Hohn et al. Retrovirology 6:92 2009; FJM van Kuppeveld et al., BMJ 340:c1018, 2010). Fischer et al. found 1 of 105 prostate cancer patients and 1 of 70 control subjects to be XMRV positive in a German study. Hohn et al. screened 589 prostate cancer patients in Germany without detecting a single positive. Van Kuppeveld et al. also failed to detect any DNA or RNA positives in 32 chronic fatigue patients or in 42 matched controls in Holland. Recently another paper from Fischer et al. (Emerg Infect Dis. 2010) showed about 10% positivity in RNA derived from sputum of 162 immunosuppressed patients and 2-3% positivity in sputum RNA from 168 normal patients in a German study. The assays did not appear to be different and no explanation was offered for the discrepancies.
The inconsistency of results calls into question the reliability of the current testing methods, in particular in DNA amplification. Following the lead of Lombardi et al. all investigators so far have used nested PCR using XMRV sequence based primers, followed by running the sample on a gel and looking for a visible band. This method is known to be variable in sensitivity and depend on the quality of nucleic acid samples. Detection of XMRV RNA has also been described mainly using the method of Dong et al. PNAS 2007, based on that of Urisman et al. 2006. In this assay RNA is prepared from tissue and/or blood, reverse transcribed to cDNA and the cDNA examined by QPCR with XMRV specific primers. As noted this led to inconsistent results (Enserink et al., Science 329:18-19, 2010). Claims of various sensitivities have been made for such tests, but it is not possible to verify any of these and the assays appear to be incompletely characterized.
A PCR based diagnostic screening assay for XMRV in human blood has been recently developed (www[.]vipdx.com), using nested PCR and gel detection of the amplification product (Lombardi et al.), with an estimated sensitivity for the nested DNA PCR around 600 proviral copies/test. In addition the report of Lombardi et al. do not show complete concordance of gag and env detection, with positives in gag and negatives for env observed in some subjects. This was attributed to variability in the assay. In all of the assays developed so far great care has been taken to use primers that will differentiate MLV from XMRV, so that only XMRV is detected. Therefore there is a great need for a reliable and validated assay for XMRV DNA and RNA in accessible samples from volunteers or patients in order to determine the real frequencies of positivity and whether there is linkage to disease. In addition a reliable blood screening assay is not available. Recent data suggest that detection of XMRV in many cases is caused by artifacts (Paprotka T., Science, 333, 97-101, 2011) or contamination with mouse DNA (Robinson M J. et al., Retrovirology, 7:108 doi: 10.1186/1742-4690-7-108, 2010).
Furthermore, gene therapy vectors based upon MLV are being used including replication competent MLV-based vectors. For example, a replicating retrovirus based on amphotropic MLV and carrying an extra cytosine deaminase gene as a therapeutic agent for cancer including primary brain cancer leading to glioblastoma multiforme (GBM) (Tai et al., Mol. Ther., 12:842-851 2005; http:(//)oba.od.nih.gov/oba/RAC/meetings/Jun2009/976_Aghi.pdf; WO2010036986) having been used. An exemplary vector is being developed by Tocagen Inc. (San Diego, Calif.) and is referred to as Toca 511 (clinical trials.gov trial# NCT01156584). Subsequent to Toca 511 administration, patients are dosed with 5-fluorocytosine that is converted in situ to 5-fluorouracil, a potent anticancer compound. As the virus is generally only able to replicate in the tumor, this results in a very specific anti-cancer effect. In order to determine whether there is replication outside the tumor, for safety and/or for correlation with efficacy assays for detection of proviral DNA in the blood and MLV RNA in the plasma are needed. FDA currently requires follow-up on patients undergoing such investigational therapies with an integrating viral vector for 15 years post-treatment (Guidance for Industry—Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors: FDA Center for Biologics Evaluation and Research November 2006; http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/ucm072961.htm). Assays are needed to accomplish this and a generally accepted marker for risk of disease and disease progression in viral diseases in general and retroviral diseases in particular is the levels over time of virus in the blood or in blood cells (Gurunathan S, Habib R E, et al. Vaccine. 2009; 27:1997-2015; Low A., Okeoma C M. et al. Virology 2009; 385: 455-463). On the other hand, replication of the virus in the tumor may leak into the periphery and blood stream and so assays that monitor the appearance and levels of viral sequence in the blood as DNA or RNA can be used to determine whether there is an effective treatment and whether there is a need to modify the treatment protocol, for example to readminister the viral vector or to use adjuvants (such as steroids) that will facilitate the viral replication in the tumor.